The broad objectives of this proposal are to understand the metabolic role of one of the most abundant folate enzymes, 10-formyltetrahydrofolate dehydrogenase (FDH, ALDH1L1), and how the inhibition of its catalysis is involved in pathogenesis. Our previous studies have demonstrated that the enzyme is present in many tissues but is most abundant in liver and kidney comprising in the former up to 1.2% of the total pool of cytosolic protein. At the same time it is strongly and ubiquitously down regulate in human cancers, through a CpG island methylation in its promoter region. Moreover, the inhibition of the enzyme associated with acetaminophen overdose or alcohol consumption is believed to contribute to liver toxicity. FDH converts 10- formyltetrahydrofolate (10-fTHF) to tetrahydrofolate (THF) and CO2. Importantly, this reaction removes carbon groups from the folate pool, in the form of CO2, thus decreasing folate-dependent biosynthetic capacity. Based on this, our first hypothesis is that FDH regulates overall flow of one-carbon groups through the folate pool and is one of the intrinsic mechanisms controlling cellular proliferation. The FDH reaction is also the rate-limiting step in formate clearance. Of note, formate is a toxic metabolit normally produced in humans by several physiological pathways. Thus, our second hypothesis is that inhibition of FDH in non-proliferating hepatocytes will lead to formate toxicity. We have recently found that FDH requires the prosthetic group, 4'- phosphopantetheine (4-PP), for catalysis. This flexible long arm is covalently attached to the enzyme, transferring the reaction intermediate between two catalytic sites, and its removal/modification completely kills enzymatic activity. Accordingly, our third hypothesis is that active metabolites of acetaminophen and ethanol inhibit FDH activity by forming adducts with its prosthetic group and this inhibition contributes to liver injury. Importantly, in the previous grant cycle we have discovered a novel enzyme, mitochondrial FDH (ALDH1L2). In the present proposal we will not only pursue FDH as a research target but will also explore the significance of the entire 10-fTHF to THF pathway by adding to the equation the mitochondrial branch of the pathway. Specific aims to address our hypothesis are: (1) Solve the crystal structure of full-length apo-FDH and the 4- PP-modified holoenzyme. (2) Determine whether FDH is inhibited in vivo by covalent adduct formation at the 4- PP arm (cultured mouse and rat hepatocytes, treated by acetaminophen and ethanol, will be the model in these experiments). (3) Investigate the role of 10-formyl-THF metabolizing pathways in animal models (Aldh1l1, Aldh1l2 and double knockout mice, generated in the PI's lab, will be the model in these experiments). The importance of folate as a nutrient, its well-established role in prevention of megaloblastic anemia, vascular disease, neural tube birth defects and cancer, as well as the necessity for better understanding mechanisms of drug-induce liver injury, the critical role of mitochondria in both folate metabolism and liver injury, nd the growing body of evidence that FDH is a key metabolic regulator make these studies particularly relevant.